The extracellular matrix (ECM) plays a central regulatory role in cellular functions such as proliferation, survival, migration and differentiation. This role is influenced by the composition and the physical state of the ECM and by factors that control its homeostasis. Among those factors are the matrix degrading metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of MMPs (TIMPs). The central theme of this research proposal is the study of the ECM- dependent effect of MMP inhibitors on malignant transformation and tumor growth. The applicant hypothesizes that by preventing the proteolytic destruction of the ECM, MMP inhibitors permit the ECM to maintain a negative control on cell transformation and proliferation. This hypothesis is based on published observations from the applicant's laboratory, demonstrating that TIMP-2, a natural inhibitor of MMPs identified and characterized by us, inhibits tumor growth in vivo, and on preliminary data indicating that non-proteolyzed fibrillar collagen arrests melanoma cells in G1 - S transition and upregulates the G1 cyclin inhibitor p27 kip1. The proposal has 3 specific aims. Specific Aim #1 will examine the mechanisms involved in the upregulation of p27 kip1 by fibrillar collagen with particular emphasis on the role of the cytoskeleton and collagen-binding integrins. They will determine whether inhibition of collagen degradation by TIMP-2 can maintain growth arrest and elevated p27 kip1 levels in vitro and in vivo. Specific Aim #2 will examine the effect of gain or loss of TIMP-2 function on tumor growth. They will develop transgenic mice in which TIMP-2 is overexpressed in a tissue specific or ubiquitous manner and use TIMP-2 knock-out mice in mammary tumor and melanoma tumor models. Specific Aim #3 will focus on malignant transformation. They will use a mammary tumorigenesis model in Wnt10b transgenic mice in which they have observed an induction of MMP-9 during tumor formation to examine in vitro and in vivo whether inhibition of MMP-9 activity by TIMP-2 can prevent malignant transformation and tumor formation. From these experiments a fundamental understanding of how the ECM affects tumor take and growth will be gained and valuable information in the design and appropriate clinical use of MMP inhibitors will be obtained.